The present invention relates to methods of transforming red microalgae and, more particularly, to red microalgae expressing exogenous polypeptides and to methods of producing and utilizing same.
The rapidly growing biotechnology industry is hampered by a limited capacity to produce recombinant polypeptides on a large scale, safely and cost-effectively (Garber, K., Nature Biotechnology 19:184-185, 2001). While manufacturing of recombinant polypeptides can be effected in animal, bacterial, or plant cell systems, each system has major limitations.
Transgenic animal systems are, to date, expensive, require long development timelines and are associated with possible contamination with animal prions and viruses, such as HIV, foot-and-mouth disease, hepatitis etc.
Bacterial systems provide the fastest and easiest way of producing recombinant polypeptides and they are particularly suitable for the synthesis of small and simple molecules, such as insulin and human growth hormone (Goeddel et al., Nature 281:544-548, 1979; Goeddel et al., Proc. Natl. Acad. Sci. USA 76:106-110, 1979; Martial et al., Science 205:602-607, 1979). However, bacterial systems are not suitable for producing complex proteins which tend to fold incorrectly and accumulate as insoluble aggregates (known as inclusion bodies) in bacterial hosts. Several fundamental differences between prokaryotic and eukaryotic organisms account for this phenomenon, including the absence of post-translational protein modification (e.g. glycosylation) in bacteria and the lack of eukaryotic-type chaperone proteins to facilitate correct folding. In addition, production of recombinant polypeptides in bacteria is associated with a risk of endotoxin contamination.
Transgenic plants provide a promising system for producing recombinant polypeptides since they do not carry any infectious agents that are harmful to man, and are highly amenable to scaling up. Thus, in an emerging industry generically known as “molecular farming”, genetically modified plants are being used for the production of commercially valuable molecules. Among the applications that are currently being developed in molecular farming are the production of low-cost vaccines and antibodies for therapeutic and diagnostic uses, the production of copious amounts of hormones, cytokines and other bio-active molecules for the treatment of chronic or lethal diseases, the production of bio-safe substitutes for various blood components, the production of degradable plastic biopolymers, the production of unlimited amounts of processing enzymes for the food and pulp industry, the production of low-cost enzymes for waste treatments, and the production of safe bio-active molecules for the cosmetic industry (Daniell et al. Medical molecular farming: production of antibodies, biopharmaceuticals and edible vaccines in plants, Trends Plant Sci. 6:219-226, 2001; Rishi et al. Molecular Farming in Plants: A Current Perspective, J. Plant Biochemistry and Biotechnology 10:1-12, 2001; Mor, et al., Edible vaccines: a concept comes of age, Trends Microbiol. 6:449-453, 1998; and Tacket and Mason A review of oral vaccination with transgenic vegetables, Microbes Infect. 1:777-783, 1999). Yet, recombinant plants growing in open fields pose a serious risk of unintentional release of transgenes to the environment. In addition, the cost of purifying recombinant polypeptides from plant material is presently very high (Schillberg et al., Molecular farming of antibodies in plants. Naturwissenschaften 90: 145-155, 2003).
Red microalgae are attractive prospects for producing recombinant polypeptides since they grow rapidly under closed controlled conditions, pose no danger of unintentional environmental release, have a high vitamin, mineral and unsaturated fatty acid content, while the cell wall polysaccharide comprises antiviral activities (Talyshinsky et al. Cancer Cell International 2: 8, 2000; Huleihel et al., J. Appl. Phycol. 13:127-134, 2001 and U.S. application Ser. No. 10/175,830). In particular, red microalgae are advantageous hosts for producing oral pharmaceutical molecules since they can be used as delivery vehicles of the pharmaceutical molecules. Unlike the rigid microfibrilar cellulose layer typically found in the cell walls of eukaryotic algae, red microalgae cells are encapsulated within a unique complex amorphous polysaccharide (Ramus, J., J. Phycol. 8:97-111, 1972) which provides protection for recombinant polypeptides undergoing an oral route of administration. On the other hand, the unique cell wall composition creates a major obstacle for delivering an exogenous DNA into the red microalgae cells.
U.S. Pat. No. 6,027,900 teaches transforming eukaryotic algae such as Phaeodactylum tricornutum by an exogenous DNA which contains the sh ble gene as a selectable marker. However, it does not describe or suggest a procedure which is suitable for transforming red microalgae.
While reducing the present invention to practice, the present inventors have developed a transformation method which is highly suitable for transforming red microalgae. Such a method can be utilized for high throughput, cost effective and safe production of transgenic pharmaceutical and/or nutritive polypeptides and can provide protective encapsulation for their convenient and safe delivery as edible products.